The metabolism of (2-14C)-5-fluorouracil in transplantable murine colon carcinomas was studied. Fluorouracil was metabolized to nucleotides and incorporated into RNA of mouse colon carcinomas and normal tissues. No significant difference was observed in three mouse colon tumors, but the extent of incorporation of the anolog into RNA of normal colon tissue was lower than that in colon tumors. Fluorouracil phosphoribosyltransferase activity, although low, was observed to be about five times higher in mouse colon carcinomas than it was in normal colon tissue. Fluorouracil and 5-fluoro-2'-deoxyuridine, as anticipated, inhibited the incorporation of (6-3H) -2'-deoxyuridine into DNA of colon carcinomas and normal tissues. Colon and spleen tissue made a more rapid recovery of capacity for DNA synthesis than did colon tumors. In normal tissues examined the recovery from inhibition of DNA synthesis by fluorodeoxyuridine appeared to be more rapid than was recovery from fluorouracil inhibition. Effects of pyrazofurin, an inhibitor of orotidylic acid decarboxylase, on pyrimidine synthesis in mouse colon carcinomas and in normal tissues was analyzed by means of high-pressure liquid chromatography. Consequences of pyrazofurin-induced inhibition of pyrimidine biosynthesis in mouse colon carcinomas were evident in decreased pools of pyrimidine ribonucleotides and in elevated levels of orotic acid and orotidine in acid-soluble extracts. The maximum reduction in uracil ribonucleotide pools was observed 24 hours after pyrazofurin treatment. Recovery of uracil ribonucleotide pools was evident within 48 hours and was complete 72 hours after treatment. The maximum level of orotic acid-orotidine in colon carcinomas was attained 24 hours after treatment; these levels remained elevated above control levels 72 hours after pyrazofurin treatment.